In peptide mass fingerprint map database searching, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss Prot database were performed using the Mascot search engine with a mass tol erance of 50 p. p. m. Protein scores of 56 indicate identity or extensive homology and were AEB071 1058706-32-3 considered significant. Bioinformatics analysis To do phosphorylation site prediction of the identified proteins, we used on line PhosphoSitePlus system biology resource for predicting the presence of the phosphorylation modification sites, and PubMed database searching for comparing with the phos phorylated proteins reported in the literature.

In addition, KEGG pathway analysis of the identified proteins was done in DAVID bioinformatics resources. Validation of EGFR signaling phosphoproteins by IP Western blotting Cells were lysed in the lysis buffer containing 150 mM NaCl, 50 mM Tris HCl, 1 mM EDTA, 1% Tti ton X 100, 1% NP 40 supplemented with phosphatase inhibitor cocktail 1 and 2 at 4 C, and subsequently centrifuged AG-014699 PARP 阻害剤 at 12000 g for 30 min at 4 C. Total cellular proteins were immunoprecipitated with protein G Sepharose, and anti target protein antibody overnight at 4 C. Immunocom plexes were used for Western blotting. Briefly, proteins were separated by 7% SDS PAGE, and transferred to a PVDF membrane. Blots were blocked with 3% BSA for 1 h at room temperature, and then incubated with pri mary antibody, followed by incubation with HRP conju gated secondary antibodies for 1 h at room temperature.

The signal was visualized using ECL detection reagent. Transient transfection The cells were transfected with GSTP1 siRNA or con trol siRNA according to the siRNA transfection protocol provided by the manufac turer. Briefly, the day before transfection, CNE2 cells were plated into 6 well plates at the density of 105 cells mL in DMEM medium containing 10% FBS. When the cells were 60 80% confluent, they were trans fected with 10 nmolL of GSTP1 siRNA or control siRNA in serum free DMEM medium using Lipofecta mine 2000 reagent. 4 h after the beginning of the transfection, the medium was replaced with DMEM medium containing 10% FCS, and continued to culture the cells for additional 44 h, and then GSTP1 expression level was determined by Western blotting.